The objective of the project is to examine the hypothesis that cellular differentiation and malignant transformation are accompanied by expression of specific cell surface proteins. The surface proteins will be analyzed by a newly developed two-dimensional gel electrophoresis system. The final goal of the proposal during the period of grant support is to analyze and compare surface proteins of the normal and T/t locus mutant mouse embryos. The recessive t mutants of homozygous lethal type are uniquely suited for this project since: 1. they interfere with development of embyros at six specific stages, 2. they interfere with the further differentiation of ectoderm, 3. they are expressed as the surface antigens, and 4. a number of mutant strains are available. I propose to analyze proteins of sperm, unfertilized eggs, fertilized eggs and embryos. Analysis of sperm proteins is feasible by techniques available in this laboratory. However, sensitivity of protein detection needs to be improved for the analysis of proteins in eggs and embryos. An approximately 10 to the 4th power-fold increase of sensitivity seems possible by utilizing horseradish peroxidase-catalyzed reaction as follows: Proteins are tagged with biotin by reacting with succinimidated biotin. Biotin molecules are then complexed with avidin-horseradish peroxidase conjugate. Finally, horseradish peroxidase will be detected by catalytic polymerization of a radioactive substrate such as tyrosine, aniline derivatives or diaminobenzidine or its derivatives. A cell surface protein of 135,000 molecular weight is inducible in Chinese hamster ovary cells by cyclic AMP derivatives or prostaglandin E1. This is an ideal model system to study rate of synthesis and turnover of surface proteins, pathway of protein expression on cell surfaces, and mode of protein-protein interaction on plasma membrane.